InputType: | NuclearExtractsorPurifiedEnzymes |
ResearchArea: | RNAMethylation |
TargetApplication: | AmountQuantitation |
VesselFormat: | 96-WellPlate |
100%Guarantee: | 6months |
The Epigenase™m6ADemethylaseActivity/InhibitionAssayKit(Colorimetric) isacompletesetofoptimizedbuffersandreagentstocolorimetricallymeasuretheactivity/inhibitionoftotalm6Ademethylasesusingnuclearextractsorpurifiedm6AdemethylaseslikeFTOandALKBH5fromabroadrangeofspeciessuchasmammalian,plant,fungal,andbacterial,inavarietyofformsincluding,butnotlimitedto,culturedcellsand,freshandfrozentissues.Thiskithasthefollowingadvantagesandfeatures:
BackgroundInformation
N6-methyladenosine(m6A)isthemostcommonandabundantmodificationonRNAmoleculespresentineukaryotes.Recently,DNAm6AisalsoidentifiedinmulticellulareukaryotesincludingCaenorhaBDitiselegansandDrosophilamelanogaster,andfurThermoreidentifiedinhighereukaryotesincludingplants,mouseandhumancells.m6AplayscrucialrolesinregulatingDNAreplication,DNAdamage,RNAsplicing,transposition,transcription,andcellulardefense.Inhumancells,them6AmodificationisprobablycatalyzedbyamethyltransferasecomplexMETTL3/METTL14andremovedbytheα-ketoglutarate(α-KG)-andFe2+-dependentdioxygenasessuchasFTO,ALKBH5andTET-likeenzymes.ItwasshownthatMETTL3andα-KG/Fe2+-dependentdioxygenasesplayimportantrolesinmanyBIOLOGicalprocesses,rangingfromdevelopmentandmetabolismtofertility.Thedynamicandreversiblechemicalm6AmodificationonDNA/RNAmayalsoserveasanovelepigeneticMarkerofprofoundbiologicalsignificance.Down-regulationofm6Amodificationwasfirstcharacterizedinhumancancercellsandtissues,relativetotheirnormalcontrols.m6AisfoundtobethemostregulatedDNAmodificationincancers.Inadditiontotheregulationincancercells,relativetotheprimarycell/tissueswhichcontainquitelowDNAm6A(<0.001%),ahundreds-foldincreaseofm6Amodificationwasfoundforinvitroculturedhumancells(0.03%-0.22%).
Principle&Procedure
Thiskitisdesignedformeasuringtotalm6Ademethylaseactivity/inhibition.Inanassaywiththiskit,theuniquem6Asubstrateisstablycoatedonthestripwells.Activem6Ademethylasesbindtoanddemethylatem6Acontainedinthesubstrate.Theun-demethylatedm6Ainthesubstratecanberecognizedwithahighaffinitym6Aantibodyandtheimmuno-signalisenhancedwithenhancersolution.Theratiooramountofun-demethylatedm6A,whichisinverselyproportionaltoenzymeactivity,canthenbecolorimetricallyquantifiedthroughanELISA-likereaction.
StartingMaterials
Inputmaterialscanbenuclearextractsorpurifiedenzymes.Theamountofnuclearextractsforeachassaycanbe2µgto20µgwithanoptimalrangeof5µgto10µg.Theamountofpurifiedm6ADNAdemethylasescanbe20ngto1µgwithanoptimalrangeof50ngto500ng,dependingonthepurityandcatalyticactivityoftheenzymes.
WB(10XWashBuffer)
DB(DemethylaseBuffer)
MS(10Xm6ASubstrate)*
BS(BindingSolution)
AS(AssayStandard,2µg/ml)*
CA(CaptureAntibody,1000µg/ml)*
DA(DetectionAntibody,400µg/ml)*
ES(EnhancerSolution)*
DS(DeveloperSolution)
SS(StopSolution)
Co-Factor1*
Co-Factor2*
Co-Factor3*
8-WellAssayStrips(WithFrame)
*Formaximumrecoveryoftheproducts,centrifugetheoriginalvialpriortoopeningthecap.
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